JP2013527157A - Prodrug composition, prodrug nanoparticles and methods of use thereof - Google Patents

Abstract

A nanoparticle comprising a prodrug and a prodrug linked to a phospholipid, wherein the linkage promotes release of the prodrug from the nanoparticle to a target cell or site in the cell membrane by fusion of the particle and cell membrane Nanoparticles are disclosed. Further disclosed are methods of making and using the nanoparticles and their components.

Description

  The present invention includes prodrug compositions, nanoparticles comprising one or more prodrugs, and methods of use thereof.

Government Rights This invention was made with government support under NS059302 and CA119342 awarded by the National Institutes of Health. The government has certain rights in the invention.

  Known active compounds with in vitro activity may not be accessible to in vivo therapies due to obstacles in the delivery of these compounds. For example, a compound may have a short half-life in vivo, making the compound actually useless in vivo. Similarly, compounds can be unstable in vivo and can turn into inactive compounds under physiological conditions. Alternatively, the compound may be associated with significant toxicity, making its in vivo use difficult. Accordingly, there is a need for a method of delivering these active compounds so that the activity of the compounds is maintained in vivo, avoiding potential toxicity and reaching the desired target cells.

  One embodiment of the present invention includes a composition for in vivo delivery of a compound to a target cell. The composition comprises non-liposomal particles and at least one prodrug. The outer surface of the particle is a membrane composed of at least one prodrug, further composed of about 100% to about 60% phospholipids, and the outer surface of the particle is in vivo on the leaflets outside the cell membrane of the target cell. Fusions can result in a prodrug transfer from the particle to the outer leaflet of the target cell. Prodrugs comprise a compound of less than about 3000 da linked to the acyl moiety of a phosphoglyceride, where the compound may be released (or released: released) from the phosphoglyceride backbone by enzymatic cleavage. The prodrug essentially remains on the outer surface cell membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell, where the prodrug is further transported from the outer leaflet to the inner leaflet of the target cell's cell membrane The cleavage of the cleavable linkage (linkage) results in the release of the compound intracellularly.

  Another aspect of the invention encompasses a method for in vivo delivery of a compound to a target cell. The method comprises administering to the subject a composition, ie, a composition comprising non-liposomal particles and at least one prodrug in a pharmaceutically acceptable carrier. The outer surface of the particle is a membrane composed of at least one prodrug, further composed of about 100% to about 60% phospholipids, and the outer surface of the particle is in vivo on the leaflets outside the cell membrane of the target cell. Fusions can result in a prodrug transfer from the particle to the outer leaflet of the target cell. Prodrugs comprise a compound of less than about 3000 da linked to the acyl moiety of a phosphoglyceride, where the compound may be released from the phosphoglyceride backbone by enzymatic cleavage. The prodrug remains essentially on the outer surface cell membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell, and the prodrug is further transported from the outer leaflet to the inner leaflet of the target cell's cell membrane. The cleavage of the enzyme-cleaving linkage results in the release of the compound intracellularly.

  Other aspects and iterations of the invention are discussed in more detail below.

The application document referring to the color drawing contains at least one photograph taken in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 shows a light microscopic image of cells treated with doxorubicin (A) and a free doxorubicin drug (B) delivered by a prodrug.

FIG. 2 is a graph showing the effect of free doxorubicin drug and prodrug doxorubicin on cell proliferation compared to untreated control (control).

FIG. 3 is a graph depicting the angiogenic response to prodrugs of doxorubicin and paclitaxel incorporated into integrin-targeted PFC nanoparticles in a rat matrigel plug model (n = 2 / group). ).

Figure 4 shows nonsense changes in angiogenic contrast enhancement at 1.5T with non-prodrug paclitaxel (A) and integrin in the vx2 tumor model (n = 2 / group) in rabbits. 2 depicts two graphs illustrating the angiogenic response (B) to paclitaxel prodrug incorporated into PFC nanoparticles.

FIG. 5 represents the sites available for doxorubicin and functional modifications.

FIG. 6 represents a synthesis scheme of PC-doxorubicin conjugate 1.

FIG. 7 represents ¹ H NMR (400 MHZ) spectroscopy of PC-doxorubicin (PC-DXR) candidate-1 prodrug and its prodrug.

FIG. 8 represents the synthesis scheme of taxol prodrug.

FIG. 9 is a graph showing the antiproliferative effect of PC-paclitaxel (PC-PXTL) prodrug on 2F2B endothelial cells.

FIG. 10 represents a synthetic scheme for cisplatin-prodrug-1.

FIG. 11 represents a synthetic scheme for cisplatin-prodrug-2.

FIG. 12 represents a synthetic scheme for methotrexate prodrugs.

FIG. 13 represents a synthesis scheme for the brotezomib prodrug.

FIG. 14 represents a synthetic scheme for myc-inhibitor prodrug (A) and alternative variant (B).

FIG. 15 represents a synthetic scheme for a lenalidomide prodrug.

FIG. 16 represents a synthesis scheme of fumagillin prodrug 1 (top) and fumagillin prodrug 2 (bottom).

FIG. 17 represents a synthetic scheme for photodynamic therapy (PDT) prodrugs.

FIG. 18 represents the synthetic scheme of docetaxel-prodrug (A) and alternative variant (B).

FIG. 19 presents two graphs illustrating that fumagillin dissolved in a lipid membrane is stable in vitro but is rapidly lost in vivo. (A) Fumagillin in vitro release is maintained for 4 days. (B) In vivo release of fumagillin occurs within minutes.

FIG. 20 depicts a graph showing that administration of fumagillin prodrug targeted to αvβ3 reduces transplant tissue volume increase.

Figure 21 shows that fumagillin prodrug targeted to αvβ3 reduces cell proliferation (A) as measured by CyQuant assay, and by Alamar blue assay in HUVEC cells. 2 represents two graphs illustrating the metabolic activity (B) of a cell as measured.

FIG. 22 presents a graph illustrating the results of Alamar's blue assay with docetacel prodrug. 7,000 2F2B cells / well were exposed to particles comprising either the drug pear (control), docetacel or docetacel prodrug targeted to αvβ3.

FIG. 23 presents micrographs and graphs that demonstrate the effect of docetacel prodrug on tumor growth. (A) Four panels were exposed to either αvβ3 targeted particles comprising baseline and docetacel prodrugs or αvβ3 targeted particles without docetacel 3 A micrograph of the tissue after time injection is shown. (B) Tumor volume decreases with docetacel prodrug administration. (C) Illustrative tumor volume with particles targeted to αvβ3 (left panel) or particles targeted to αvβ3 without docetacel (right panel) comprising a docetacel prodrug.

FIG. 24 represents the effect of docetacel prodrug on the proliferation index. (A) Particles targeted to αvβ3 and comprising a docetacel prodrug reduce the proliferation index of tumor cells. (B) The micrograph demonstrates reduced growth after exposure to αvβ3-targeted particles comprising docetacel. The proliferation index is equal to the PCNA positive area divided by the methyl green nucleus single positive area.

FIG. 25 represents the effect of docetacel prodrug on cellular self-destruction (apoptosis) index. (A) Particles targeted to αvβ3 and comprising a docetacel prodrug reduce the cellular self-destruction index of tumor cells. (B) The micrograph demonstrates reduced cell self-destruction after exposure to αvβ3-targeted particles comprising docetacell. The cell kill index is equal to the TUNEL positive area divided by the methyl green nucleus single positive area.

FIG. 26 shows the structure of myc drug (A) and trypan blue exclusion using Trypan Blue Exclusion, cultured at different concentrations of myc-drug compared to controls containing corresponding volumes of DMSO. 2 shows a graph (B) showing the effect of the myc drug on the growth of multiple myeloma cell lines iH-929, ii.LP-1, iii.KMS-11, iv.UTMC-2.

FIG. 27 depicts a graph showing that myc-prodrug PFC nanoparticles targeted to αvβ3 reduce SMC proliferation in 48 hours.

FIG. 28 is a graph illustrating that myc-max antagonist prodrug inhibits melanoma growth in vitro at 48 hours (A) and 72 hours (B). Tg: targeted; PFC: perfluorocarbon particles; tween: polysorbate micelles.

FIG. 29 represents a diagram of polysorbate micelles.

FIG. 30 represents a tapping mode AFM image diagram of an aqueous dispersion of nanocelle droplets placed on a glass surface.

FIG. 31 represents a prodrug comprising methylprednisolone.

FIG. 32 represents a prodrug (A) comprising camptothecin and a scheme (B) for synthesizing a camptothecin prodrug.

FIG. 33 represents two prodrugs comprising PNA.

FIG. 34 represents a scheme for making fumagillin analogs.

Detailed Description of the Invention
The present invention provides one or more prodrug compositions, particles comprising one or more prodrugs and methods of use thereof. Advantageously, the present invention provides compositions and methods for in vivo delivery of compounds to target cells.

  In one embodiment, the present invention provides a drug delivery system comprising a prodrug (described in Section I below) embedded in non-liposomal nanoparticles (described in Section II below). Include. The nanoparticles may further optionally include homing ligands as described herein. The drug delivery system allows intracellular delivery of the prodrug while protecting the prodrug from physiological conditions that may inactivate the drug.

I. Prodrug Compositions Generally speaking, the prodrugs of the present invention include compounds linked to the acyl moiety of a phosphoglyceride. Suitable phosphoglycerides may include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine. The compound may be linked to one position in the phosphoglyceride. For example, the compound may be linked to the sn1 or sn2 position of the phosphoglyceride. In a preferred embodiment, the compound is linked to the sn2 position.

(a) Suitable compounds Generally speaking, suitable compounds of the invention are less than 3000 da. In one embodiment, the compound has about 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, Less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100da. In another embodiment, the compound is between about 2500 da and about 200 da. In yet another embodiment, the compound is between about 2000 da and about 200 da. In some embodiments, the compound may be greater than 3000 da so long as the prodrug comprising the compound maintains the ability to migrate from the outer leaflet of the cell membrane to the inner leaflet of the cell membrane.

  Typically suitable compounds comprise reactive groups outside the active site of the compound. This reactive site allows the compound to bind to the glyceride backbone as described below. Suitable compounds of the present invention are typically from the group comprising small molecules, metal atoms, organometallic complexes, radioactive compounds, amino acid polymers (eg proteins, peptides, etc.), carbohydrates, lipids, nucleic acid polymers or combinations thereof. May be chosen. Generally speaking, suitable compounds may be linked to phosphoglycerides as described herein. Once the compound is cleaved from the glyceride backbone, the released dosage form may be an active compound or a pre-active compound. The compound may be derivatized to enhance the desired properties of the compound. Methods for derivatizing compounds are known in the art.

  In some embodiments, for example, the compounds are drugs, therapeutic compounds, steroids, nucleic acid based materials, or derivatives, analogs or combinations thereof in their conventional dosage forms, in their original form, or to nanoparticles May be derivatized with a hydrophobic or packed portion to enhance uptake or adsorption. Such compounds may be water soluble or hydrophobic. Non-limiting examples of compounds include immune related agents, thyroid agents, respiratory products, antitumor agents, anthelmintic agents, antimalarial agents, antimitotic agents, hormones, antiprotozoal agents, antituberculosis agents, Cardiovascular products, blood products, biological response modifiers, antifungal agents, vitamins, peptides, antiallergic agents, anticoagulants, cardiovascular agents, metabolic enhancers, antiviral agents, angina It may include drugs, antibiotics, anti-inflammatory agents, anti-rheumatic agents, narcotics, cardiotonic glycosides, neuromuscular blocking agents, sedatives, local anesthetics, general anesthetics, or radioactive atoms or ions. In certain embodiments, the compound is an aptamer or nucleic acid derivative, such as a peptide nucleic acid (PNA) or a locked nucleic acid (LNA).

  In an exemplary embodiment, the compound has doxorubicin, paclitaxel, taxol, lenalidomide, methotrezate, bortezomib, myc-inhibitor, lenalidomide, cisplatin, methylprednisolone, fumagillin, camptothecin, peptide nucleic acid, PDT drug, or pharmaceutical activity. It may be a derivative or analog thereof that retains it. In one embodiment, the compound may be an analogue of fumagillin. In some embodiments, the fumagillin analog is selected from the analogs detailed in Example 12. In other embodiments, the compound may be a PDT drug (prodrug of formula XI). Additional suitable PDT drugs are known in the art and may include porfimer sodium, aminolevulinic acid, and the methyl ester of aminolevulinic acid.

In other exemplary embodiments, the compound may be selected from the compounds listed in Table A below.
Table A: Non-limiting examples of compounds

(b) Linkage
As defined in section I (a) above, the prodrugs of the present invention include compounds linked to phosphoglycerides. The linkage between the glyceride backbone and the compound may be any suitable linkage known in the art. In typical embodiments, suitable linkages may include carbon, oxygen, nitrogen or combinations thereof.

  Linkage is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or counting from the glyceride backbone It may contain 20 or more atoms. In an exemplary embodiment, the linkage comprises at least 5 atoms, counted from the glyceride backbone.

  Generally speaking, the linkage is chosen such that the sn2 position of the glyceride backbone is cleaved by the enzyme. Typically, such enzymes may be found in the cell.

  Furthermore, the linkage may itself be enzyme cleavable. In an exemplary embodiment, the linkage may be cleaved by intracellular enzymes. In some embodiments, either the glyceride sn2 position or the linkage may be cleaved by a lipase. For example, in one embodiment, the lipase may be a phospholipid A2 lipase (also known as phospholipase A2). In another embodiment, the lipase may be a phospholipid A1 lipase (also known as phospholipase A1). In yet another embodiment, the lipase may be a phospholipase B enzyme. In each of the above embodiments, the enzyme can cleave the linkage and consequently release the compound from the linkage.

(c) Released Form of Compound When the prodrug of the present invention is cleaved from the glyceride backbone, a released form of the compound is produced. The released form may be the active compound or the pre-active compound. As used herein, an “active compound” is a compound that has a pharmaceutical effect on a cell. Such active compounds may comprise a linkage as defined in section I (b) above, or a portion thereof. An “active compound” may further be a metabolite of a compound used to produce a prodrug.

  A pre-active compound, as used herein, is a compound that requires an enzyme activator to become an active compound as defined above. For example, the pre-active compound of the present invention may comprise a compound defined in section I (a) above, a linkage defined in section I (b) above, or a portion thereof. The linkage may include an enzyme recognition site within the linkage or between the linkage and the compound. Generally speaking, an enzyme binds to an enzyme recognition site and cleaves the link. It removes the linkage (or part thereof) from the pre-active compound to yield the active compound, as defined above. In other embodiments, the active compound may be a metabolite of the pre-active compound.

(d) Nanoparticles comprising a prodrug In an exemplary embodiment of the application, the prodrug is contained in a nanoparticle. In such embodiments, the prodrug is not substantially released from the nanoparticle until after fusion of the target cell's cell membrane with the nanoparticle. In an exemplary embodiment, the prodrug is not substantially released from the nanoparticle until after fusion of the nanoparticle with the outer leaflet of the cell membrane of the target cell.
Such a fusion is described in more detail below. Briefly, however, the fusion of the target cell's cell membrane and nanoparticles allows lipid transfer between the nanoparticles and the target cell's cell membrane. For example, in some instances, the lipid membrane of the nanoparticle may be fused to a leaflet external to the lipid membrane of the target cell's cell membrane, allowing the transfer of the prodrug to the nanoparticle-derived lipid and / or the target cell's cell membrane. to enable.

Following fusion and subsequent transfer of the prodrug from the nanoparticle to the target cell, the prodrug may be cleaved, separating the released form of the compound from the glyceride backbone. Generally speaking, this cleavage occurs after the transition of the prodrug from the outer leaflet to the inner leaflet of the target cell's cell membrane. This results in compounds released substantially intracellularly. The released compound may contain a linkage or part thereof as described above. For more details, see Section II and Examples below.
(e) Typical embodiment

  In exemplary embodiments, the prodrugs of the present invention have the formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII Or a prodrug of XIX. In another exemplary embodiment, the prodrug of the present invention has the formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII A prodrug consisting of XVIII or XIX. In another exemplary embodiment, the prodrug of the present invention has the formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII , XVIII or XIX prodrugs, wherein the compound is further derivatized so as not to significantly alter the function of the prodrug. In another more exemplary embodiment, the prodrug of the present invention has the formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, Comprising a prodrug of XVII, XVIII or XIX, wherein the phosphoglyceride backbone is further derivatized so as not to significantly alter the function of the prodrug.

In each of the above exemplary embodiments, a compound of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX Prodrugs may have linkages between the glyceride backbone and the compounds that may differ in the number of atoms represented in the formula. For example, the linkage is 1, 2, 3 for formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX. , 4, 5, 6, 7 or more than 7 atoms in length.
Table B: Typical prodrugs

II. Nanoparticles The nanoparticles of the present invention comprise a prodrug and an amphiphilic membrane as discussed in Section I above. In some embodiments, the amphiphilic membrane comprises a prodrug. The membrane is stable in vivo for at least the time required to fuse to the cell membrane of the target cell for the purpose of particles and to transfer the prodrug to the cell membrane of the target cell.

  Advantageously, the nanoparticles of the present invention can protect highly labile compounds from inactivation in vivo. This is due to the sequestration of the compound, partly in the form of a prodrug, in an amphiphilic membrane. The membrane protects the compound from hydrolysis and inactivation due to physiological conditions (eg pH or enzymatic cleavage). This extends the half-life of the compound in vivo, allowing delivery of the active substance to the cell, thereby enhancing the pharmacokinetic and pharmacodynamic properties of the compound. As a result, compounds that have been previously formulated as prodrugs and incorporated into the nanoparticles described herein that have previously had little in vivo efficacy can be viable in vivo therapeutic options.

  In general, the nanoparticles of the present invention may be from about 10 nm to about 10 μm. In some embodiments, the nanoparticles are about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200. 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450 , 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700 , 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950 960, 970, 980, 990 or 1000 nm. In other embodiments, the nanoparticles are about 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75. Or it may be 6 μm. In yet another embodiment, the nanoparticles of the present invention may be less than 100 nm. In yet another embodiment, the nanoparticles can be less than 200 nm.

(a) Nanoparticle Amphiphilic Film The nanoparticle of the present invention comprises an amphiphilic film. Generally speaking, the amphiphilic membrane may be comprised between 100% and 50% of the amphiphilic lipid (eg, phospholipid). In some embodiments, the membrane is about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, It is composed of 55, 54, 53, 52, 51 or 50% phospholipids.

  The composition of the amphiphilic membrane may vary, but is typically comprised between about 0.1% to about 75% of the prodrugs discussed in Section I above. In one embodiment, the lipid membrane of the nanoparticle comprises about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1% of the prodrug. In another embodiment, the lipid membrane of the nanoparticles is a prodrug of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 68, 69, 70, 71, 72, 73, 74 or 75 percent. In some embodiments, the nanoparticulate lipid membrane comprises from about 10% to about 60% of the prodrug. In other embodiments, the lipid membrane of the nanoparticles comprises from about 20% to about 50% of the prodrug. In yet other embodiments, the lipid membrane of the nanoparticles comprises from about 30% to about 40% of the prodrug.

  The nanoparticulate lipid membrane may include one or more prodrug types. For example, the nanoparticulate lipid membrane may comprise 1, 2, 3, 4, 5, 6, or 6 or more types of prodrugs. In each example, the sum of the percent of the membrane comprising the prodrug typically will be from about 0.1% to about 70%.

  Typically, a stable membrane of nanoparticles of the present invention must be compatible with in vivo use. For example, nanoparticulate lipid membranes should not undergo substantial complement activation pathways or hemolysis.

  Until the prodrug is substantially released from the particle until after the fusion of the target cell membrane with the particle, the composition of the stable membrane may vary. The prodrug can then migrate from the nanoparticle lipid membrane to the target cell membrane. The amphiphilic membrane may comprise a polymer or a combination of lipids and polymers. In one embodiment, the stable membrane comprises an amphiphilic material. The phrase “amphiphilic material” as used herein refers to a material that has both hydrophobic and hydrophilic moieties (eg, lipid materials or amphiphilic polymers). The amphiphilic material may be a natural, synthetic or semi-synthetic material. As used herein, “natural” refers to materials that may be found in nature, “synthetic” refers to materials that may be produced in a laboratory setting, and “semi- “Synthetic” refers to natural materials that have been modified in laboratory settings. In one embodiment, the amphiphilic material is a lipid material. For example, the outer layer may be a single lipid layer or may include multiple lipid layers. Lipid material is used herein in its broadest sense, derived or natural or synthetic phospholipids, fatty acids, cholesterol, lysolipids, lipids, sphingomyelin, tocopherols, glucolipids, stearylamines, cardiolipin, plasmalogens, Ethers are functionalized with lipids with fatty acids linked to esters, polymerized lipids, lipoproteins, glycolipids, derivatized surfactants, drug functionalized lipids, and targeted ligands. Lipids, contrast agent complex lipids, lipid polymers, surfactants or combinations thereof.

  The amphiphilic membrane may further comprise polyethylene glycol bound to lipid (PEG). Generally speaking, however, the membrane should contain no more than 10% PEG. In some embodiments, the membrane comprises less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% PEG.

  In addition, the stable membrane may contain a surfactant. In some embodiments, preferred surfactants are phospholipids and cholesterol. In addition, the surfactant may include but is not limited to: 1,2-dipalmitoyl-sn glycerin-3-phosphoethanolamine-N-4- (p-maleimidophenyl) butyramide, amine- PEG2000-phosphatidylethanolamine, phosphatidylethanolamine, gum arabic, cholesterol, diethanolamine, glyceryl lanolin monostearate, lecithin, egg yolk lecithin mono- and diglyceride monoethanolamine oleic acid, oleyl alcohol, poloxamer, peanut oil, palmitic acid, poly Oxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, poly Sorbate 80, propylene glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, stearic acid, trolamine and emulsifying wax. The above surfactants may be used alone or in combination to help stabilize the nanoparticles.

  In one embodiment, the amphiphilic membrane of the particles may include polysorbate. For example, the membrane may comprise between about 1 and about 50% polysorbate. In certain embodiments, the membrane is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, It may contain 47, 48, 49 or 50% polysorbate.

  In addition, suspending agents or viscosity increasing agents that may be used include but are not limited to: gum arabic, agar, alginic acid, aluminum monostearate, bentonite, magma, carbomer 934P, carboxymethylcellulose, calcium And sodium and sodium 12, carrageenan, cellulose, dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, magnesium aluminum silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide, sodium alginate , Tragacanth and xanthan gum.

  In various embodiments, the lipid membrane amphiphilic material may be cross-linked to stabilize the nanoparticles. However, such cross-linking should further allow appropriate lipid mobility to facilitate prodrug transfer from the nanoparticle to the cell membrane of the target cell. In some embodiments, the particles may be crosslinked on the surface of the outer layer. In other embodiments, the particles may be cross-linked within the outer layer. The cross-linking may be chemical cross-linking or photochemical cross-linking. Briefly, a suitable cross-linking agent reacts with one or more active groups in the outer layer. The crosslinker may be homobifunctional or heterobifunctional. Suitable chemical crosslinkers may include glutaraldehyde, biscarboxylic acid spacers or biscarboxylic acid active esters. Furthermore, the outer layer may be chemically cross-linked using a bislinkeramine / acid via a carbodiimide coupling protocol. Alternatively, the particles may be cross-linked using a click chemistry protocol. In yet another embodiment, carbodiimide coupling chemistry, acylation, active ester coupling or alkylation may be used to crosslink with the outer layer. In an exemplary embodiment, the cross-linking is carbodiimide mediated. In some embodiments, EDC (also EDAC or EDCI, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, ie, a highly water soluble carbodiimide, is coupled to a primary amine that provides an amide bond. Adopted in pH range of 4.0-6.0 to activate carboxyl group for use EDC is used in combination with N-hydroxysuccinimide (NHS) or sulfo-NHS to enhance coupling efficiency. Those skilled in the art will recognize that the appropriate cross-linking agent may and will vary depending on the particle and the composition of intended use.

(b) fusion
The nanoparticles of the present invention may be fused to the target cell membrane. As used herein, “fuse” refers to the alignment of the lipid membrane of the target cell and the lipid membrane of the nanoparticle. It is such that an interaction between the two membranes may occur. In an exemplary embodiment, “fusion” refers to a hemifusion of particulate cell membranes with leaflets outside the cell membrane of the target cell. (Lanza, et al. Circulation 2002; 106: 2842; Partlow et al. Biomaterials 2008; 29: 3367; Soman, et al. Nano Letters 2008; 8: 1131-1136). In one embodiment, the nanoparticles of the present invention are stable in vivo for a time required to fuse to the cell membrane of the target cell and transport the prodrug to the cell membrane of the target cell, at least for the purpose of the nanoparticle. Comprising a lipid membrane. In other words, the nanoparticles of the present invention substantially retain the prodrug within the lipid membrane of the nanoparticle until the nanoparticle is fused to the target cell membrane and does not transfer the prodrug from the nanoparticle to the target cell membrane. To do. In another embodiment, the nanoparticle of the present invention is a lipid membrane that is fused in the cell membrane of a target cell for the purpose of at least the nanoparticle and that is stable in vivo for the time required for the eating and drinking action of the nanoparticle. Comprising.

  The length of time required to fuse the nanoparticle to the target cell membrane for purposes is in addition to other factors, the nanoparticle (e.g., it comprises a homing ligand), the size of the nanoparticle, Depending on the intended target cell location and the prodrug utilized, it can vary. One skilled in the art can perform analyzes known in the art to determine the ideal length of time required for the particle nanoparticle / prodrug / target cell combination.

  In some embodiments, the length of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 Or 20 minutes. In other embodiments, the length of time is more than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 30 minutes. For example, the length of time may be greater than about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 100 minutes. In an exemplary embodiment, the length of time is approximately 20 minutes.

(c) Prodrug transfer As briefly mentioned above, the prodrug of the present invention may be transferred from the lipid membrane of the nanoparticle to the lipid membrane of the cell. In certain embodiments, the lipid membrane of the nanoparticles must be mobile in order to allow transport of the prodrug. Furthermore, in certain embodiments, the lipid of the nanoparticulate lipid membrane must be able to freely exchange with the lipid membrane of the target cell. In other words, in these embodiments, the nanoparticulate lipid membrane should not have an intrinsic surface barrier or energy barrier to lipid exchange with the target cell membrane. For example, the lipid composition of the nanoparticulate lipid membrane may be adhered. Lipid compositions were tested using methods known in the art to ensure proper lipid mobility for transporting prodrugs from nanoparticles to target cell membranes. In an exemplary embodiment, as shown in the Examples, the membrane of particles forms a hemifusion structure with leaflets outside the target cell membrane. As shown in the examples, such a fusion event may be visualized. Thus, one skilled in the art can determine whether a special membrane formed using the guidelines detailed above forms a hemifusion structure with a target cell membrane.

  The amount of time required to transfer the nanoparticle to the lipid membrane of the target cell, in addition to other factors, is determined by the nanoparticle (e.g., it comprises a homing ligand), the size of the nanoparticle, Depending on several factors including the intended target cell location and the prodrug utilized, it can and will vary. In some embodiments, the length of time is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 Or 20 minutes. In other embodiments, the length of time is more than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 30 minutes. For example, the length of time may be greater than about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 100 minutes.

(d) Homing Ligand In some embodiments, the nanoparticles of the present invention may comprise a homing ligand. As used herein, “homing ligand” refers to a biomolecule that assists in the fusion of a target cell membrane and a nanoparticle.

  The homing ligand may comprise antibodies, antibody fragments, proteins, peptides, carbohydrates, lipids, small molecules, polysaccharides, nucleic acids, aptamers, peptidomimetics, other mimetics and pharmaceuticals, alone or in combination ( But not limited to these). Furthermore, the homing ligand may comprise a microorganism (phage or virus). The homing ligand may further be a designed analog or derivative of each of the above. The homing ligand may be attached directly or indirectly to the nanoparticle.

  Direct conjugation of a homing ligand to a nanoparticle refers to the preparation of a homing ligand-particle complex. There, homing ligands are adsorbed by ion, electrostatic, hydrophobic or other non-covalent means to the nanoparticle surface (e.g. between acylated antibodies or complementary nucleic acid sequences). Hybridisation), or chemically linked to the surface by covalent bonds to lipid surface components, or essentially incorporated into lipid membranes as components of membranes (e.g. lipids derived from peptidomimetic agents). .

  Indirect bonding refers to the formation of a complex of nanoparticles and homing ligand in vivo in two or more steps. Indirect conjugation utilizes a chemical binding system to provide a tight and special fusion of particles to the surface of a target cell or tissue. A non-limiting example of an indirect homing system is avidin biotin.

The avidin-biotin interaction has been incorporated into many biological and analytical systems and is a useful non-covalent homing system selected for in vivo applications.
Avidin has a high affinity for biotin (10-15M) that promotes rapid and stable binding under physiological conditions. A homing system utilizing this approach is administered in two or three steps, depending on the formulation. Typically, a biotinylated ligand (eg, a monoclonal antibody) is administered first, and “pre-homed” to a unique molecular epitope. Next, avidin is administered, which Bind to the biotin moiety of a “pre-derivatized” ligand. Finally, biotinylated particles are added and bind to unoccupied biotin binding sites, resulting in a biotinylated ligand-avidin-particle “sandwich” on avidin. The avidin-biotin approach can avoid accelerated premature clearance of particles homed induced by a mononuclear phagocyte system (MP) following the presence of surface antibodies. In addition, avidin has four independent biotin binding sites, providing signal amplification and improving detection sensitivity.

  The homing ligand may be chemically attached to the particle surface by a variety of methods depending on the homing ligand on the particle surface and the nature of the composition. Direct protein chemical conjugation of homing ligands to the particle often makes use of the numerous amino groups (eg, lysine) that are inherently present in the surface. Instead, after the particles are formed, functionally active chemical groups (e.g., pyridylthiopropionate, maleimide, or aldehyde) are incorporated into the surface as a chemistry "strap" for homing ligand conjugation. Good. Another common post-processing approach is to activate the surface carboxylate with carbodiimide prior to homing ligand addition.

  The selected covalent linkage strategy is largely determined by the chemistry of the homing ligand. For example, monoclonal antibodies and other large proteins may lose properties under severe processing conditions. However, the biological activity of carbohydrates, short peptides, aptamers, pharmaceuticals or peptidomimetics can often be preserved under these conditions.

  To ensure high binding integrity of the homing ligand and maximize nanoparticle binding activity, flexible spacer arms (e.g., polyethylene glycol, amino acids, long or short chain hydrocarbons, sugars, nucleic acids, Aptamers (eg polydextrose)) or simple caproate bridges can be inserted between the activated surface functional group and the homing ligand. These extensions may be 2 nm or more.

  The homing ligand may be immobilized within the lipid material through the use of a “primer material”. A “primer material” is any surfactant compatible compound incorporated into a particle that is chemically linked to or adsorbs to a specific binding or homing ligand. That is, incorporated into lipid membranes that may be used chemically to form covalent bonds between the particle and the homing ligand, or components of the homing ligand (if the ligand has subunits) Any element or derived element. The homing ligand may be covalently conjugated to the “primer material” with a coupling agent using methods known in the art. One type of coupling agent is carbodiimide (e.g. 1-ethyl-3- (3-N, N dimethylaminopropyl) carbodiimide hydrochloride or 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide p-toluene. Methyl sulfonate) may be used. Primer materials are amine-PEG2000-phosphatidylethanolamine, phosphatidylethanolamine, N-caproylamine phosphatidylethanolamine, N-dodecanylamine phosphatidylethanolamine, phosphatidylethanol, 1,2-diacyl-sn-glycerin-3-phospho Ethanolamine-N- [4-p-maleimidophenyl) -butyramide, N-succinyl-phosphatidylethanolamine, N-glutaryl-phosphatidylethanolamine, N-dodecanyl-phosphatidylethanolamine, N-biotinyl-phosphatidylethanolamine, N -Biotinylcaproyl-phosphatidylethanolamine and phosphatidylethylene glycol. Other suitable coupling agents include aldehyde coupling agents with one ethylene unsaturation (acrolein, methacrolein or two butenals) or multiple aldehyde groups (glutaraldehyde, propanedial or Butane dial). Other coupling agents include 2-iminothiolane hydrochloride and bifunctional N-hydroxysuccinimide ester (disuccinimidyl substrate, disuccinimidyl tartrate, bis [2- (succinimidoxycarbonyloxy) ethyl] sulfone, disuccinimidyl) Propionate and ethylene glycol bis (succinimidyl succinate) may be included Non-limiting examples of heterobifunctional reagents include N- (5-azido-2-nitrobenzoyloxy) succinimide, p-azidophenyl Bromide, p-azidophenylglyoxal, 4-fluoro-3-nitrophenylazide, N-hydroxysuccinimidyl-4-azidobenzoate, m-maleimidobenzoyl N-hydroxysuccinimide ester, methyl-4-azidophenylglyoxal, 4 -Fluoro-3-nitrophenyl azide, N-hydroxysk Nimidyl-4-azidobenzoate hydrochloride, p-nitrophenyl 2-diazo-3,3,3-trifluoropropionate, N-succinimidyl-6- (4'-azido-2'-nitrophenylamino) hexanoate Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate, succinimidyl 4- (p-maleimidophenyl) butyrate, N-succinimidyl (4-azidophenyldithio) propionate, N-succinimidyl 3- (2-pyridyldithio ) Propionate, and N- (4-azidophenylthio) phthalamide Non-limiting examples of homobifunctional reagents include 1,5-difluoro-2,4-dinitrobenzene, 4,4 ′ -Difluoro-3,3'-dinitrodiphenylsulfone, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, p-phenylenediisothiocyanate, carbonylbis (L-methionine p-nitropheny Ester, 4,4'-dithiobisphenyl azide, erythritol biscarbonate and difunctional imide ester (dimethyladipimidate hydrochloride, dimethyl subelimidate, dimethyl 3,3'-dithiobispropionimidate hydrochloride and The covalent bond formation of the specific binding species to the “primer material” can be achieved by conventional well-known reactions, eg, neutral pH with the above reagents, and 25 It can be carried out in an aqueous solution having a temperature of less than 0 ° C. for 1 hour to overnight.

  The particulate film may generally contain between 0% and about 3% homing ligand. For example, in one embodiment, the membrane may contain up to 1% of the homing ligand, 2% of the homing ligand, or up to 3% of the homing ligand.

(e) Passive homing As an alternative to nanoparticles of the invention comprising homing ligands, the nanoparticles may use “passive homing”. As used herein, “passive homing” refers to the natural tendency of nanoparticles to accumulate and thus fuse in a target cell membrane of a tissue. For example, passive homing may refer to charge interactions between a lipid membrane of a nanoparticle and a lipid membrane of a target cell. Alternatively, in other embodiments, passive homing may refer to the enhanced osmotic effect found in tumor tissue.

(f) Nanoparticle Core The core of the nanoparticle of the present invention may and may not change without closely attaching the necessary features of the lipid membrane. Generally speaking, however, the core should allow for proper lipid transfer between the nanoparticles and the cell membrane of the target cell.

(g) Imaging / Tracking Reagent The nanoparticles of the present invention contain other imaging / tracking reagents. For example, nanoparticles can be used for microscopy (e.g., fluorescence microscopy, shared focus microscopy, electron microscopy), magnetic resonance imaging, tomography, gamma and positron emission tomography (PET / CT) (SPECT Imaging / tracking reagents that may be used for / CT, planar), radiography, computed tomography (CT), spectral CT, photoacoustic tomography (PAT) or ultrasound. Imaging / tracking reagents may be detectable in situ, in vivo, in vitro and in vitro. Microscopic imaging / tracking reagents are well known in the art and may include fluorescent molecules (FITC, rhodamine and Alexafluor cyan dye). Similarly, magnetic resonance imaging molecules, radiographic imaging molecules (such as paramagnetic and superparamagnetic agents), near infrared (NIR) visual agents and ultrasound molecules are well known in the art and suitable The imaging molecule may be selected by one skilled in the art after consideration of the composition of the particle and the intended use of the particle. In certain embodiments, the outer layer may further comprise a chelating agent in which the radiometal is to be detected by radioimaging methods (PET, SPECT and related methods).

(h) Pharmaceutical Composition The present invention comprises a pharmaceutical composition further comprising the nanoparticles of the present invention or a plurality of nanoparticles. The pharmaceutical composition may be a solution, mixture or suspension of nanoparticles. In one embodiment, the pharmaceutical composition may be a solution. In other embodiments, the pharmaceutical composition may be a mixture. In other embodiments, the pharmaceutical composition may be a suspension. A non-limiting example of a suspension is a colloid.

  In some embodiments, the pharmaceutical composition may be a colloid. Generally speaking, a colloid is a suspension of powder that does not settle easily from the suspension. The colloid may be formed by microfluidization.

  In order to allow imaging and / or treatment of biological tissue, the pharmaceutical composition of particles of the invention may be administered to a subject. Suitable subjects may include (but are not limited to) mammals, amphibians, reptiles, birds, fish and insects. Non-limiting examples of mammals include humans, non-human primates and rodents.

  The pharmaceutical composition may be formulated and administered to a subject by several different means to deliver an effective amount for therapeutic and / or imaging. Such compositions are generally parenterally, intraperitoneally, intravascularly in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, additives and vehicles as desired. Or it may be administered intrapulmonary. The term parenteral drug as used herein is topical, subcutaneous, intravenous, intramuscular, intraperitoneal, intravesical, intrauterine, intraauricular, intranasal, visual, intraocular, intrapulmonary Oral, intrapharyngeal, transrectal, i urethral or transurethral, intrauterine, intravaginal, intrasternal injection or dip. Further, the term “parenteral administration” includes nebulization or aerosol administration techniques. In one embodiment, the composition may be administered as a bolus. In a preferred embodiment, the composition may be administered intravenously. Formulations of pharmaceutical compositions are described, for example, by Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975) and Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker , New York, NY (1980).

  Injectable formulations (eg, aqueous sterile injections, oily suspensions) may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable media and solvents that may be employed are water, Ringer's solution and isotonic saline. In addition, sterile, fixed oils are usually employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids (eg oleic acid) are useful in the preparation of injectables. Surfactants including dimethylacetamide, ionic and non-ionic detergents and polyethylene glycols can be used. In addition, combinations of solvents and humidifiers (discussed above) are useful. For imaging purposes, the formulation may be in the form of a biocompatible solution or suspension for parenteral application. Other adjuvants and modes of administration are well known in the pharmaceutical art.

  Those skilled in the art will appreciate that the amount and concentration of the pharmaceutical composition administered to a subject for treatment and / or imaging will depend in part on the subject and tissue being processed and / or imaged. Recognize Methods for determining the optimal amount are known in the art. And more details can be found in the examples.

  The pharmaceutical composition of the present invention may further comprise additional pharmaceutically active compounds known in the art.

III. Methods Another aspect of the invention encompasses methods of use for the prodrugs or nanoparticles of the invention. The methods of the invention may generally be used in vivo, ex vivo or in vitro.

  In one embodiment, the present invention provides a method of delivering a compound to a cell. The method comprises administering to a cell a prodrug of the present invention. In an exemplary embodiment, the method comprises administering to the cell a nanoparticle comprising a prodrug of the present invention. In a further exemplary embodiment, the method comprises administering to the cell a targeted nanoparticle comprising a prodrug of the present invention. Generally speaking, a suitable cell is any eukaryotic cell. There, leaflets outside the cell membrane can form a hemifusion structure with the cell membrane of particles.

  In another embodiment, the present invention provides a method for increasing the half-life of a compound in the blood of a subject. The method comprises administering to a subject a prodrug of the present invention. In an exemplary embodiment, the method comprises administering a nanoparticle comprising a prodrug of the present invention. Generally speaking, a suitable subject is a mammal. In one embodiment, a suitable subject may be a rodent. In another embodiment, a suitable subject may be an agricultural animal (eg, horse, cow, pig, chicken, etc.). In other embodiments, the suitable subject is a non-human primate. In yet other embodiments, the suitable subject is a human.

  In yet other embodiments, the present invention provides a method of reducing the effective dose of a compound in a subject. The method comprises administering to a subject a prodrug of the present invention. In an exemplary embodiment, the method comprises administering to the subject nanoparticles comprising a prodrug of the present invention. Suitable subjects are as defined above.

  In yet another embodiment, the present invention provides a method for controlling the release of a compound in a cell. The method comprises administering to a cell a prodrug of the present invention. In an exemplary embodiment, the method comprises administering to the cell a nanoparticle comprising a prodrug of the present invention. Suitable cells are as defined above.

  In a further embodiment, the present invention provides a method of inhibiting angiogenesis in a subject (a method comprising administering a prodrug of the present invention to a subject). Therein, the prodrug comprises an anti-angiogenic compound. In an exemplary embodiment, the method comprises administering to the subject a nanoparticle comprising a prodrug comprising an anti-angiogenic compound.

  In an exemplary embodiment, the present invention provides compounds of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX A method of administering a prodrug to a cell is included. The method comprises a nanoparticle comprising a prodrug of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII or XIX A method of administering to a cell.

  The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. However, one of ordinary skill in the art, in light of the present disclosure, will still be able to make modifications in the specific embodiments disclosed and will not have any more similar or similar results than depart from the spirit and scope of the present invention. It should be appreciated, therefore, that the matter shown or presented in the accompanying drawings should be interpreted as illustrative and not restrictive.

  The following examples illustrate various iterations of the present invention.

Example 1. Targeting and efficacy of doxorubicin prodrug
Two examples of prodrug candidates using paclitaxel or doxorubicin linked to a phosphatidylcholine backbone with a 5-carbon sn-2 acyl spacer were synthesized (see examples below). The structure of the prepared compound was characterized by ¹ H NMR (400 MHz) spectroscopy. The effects of doxorubicin prodrugs targeted to αvβ3 integrins were tested in 2F2B mouse endothelial cells induced to express integrins. Light microscopy was used to confirm doxorubicin prodrug transfer to target cells and intracellular distribution (FIG. 1). The targeted doxorubicin prodrug was distributed in the lipid membrane, throughout the cell membrane, and in the nucleus, whereas free doxorubicin drugs were found only in the nucleus. The distribution of doxorubicin in the cell membrane serves as a reservoir for doxorubicin transfer to the nucleus for more effective treatment. Enzyme-released drugs also induced significant suppression of endothelial cell proliferation at levels greater than those induced by free doxorubicin (FIG. 2).

Example 2 In vivo activity of prodrugs of doxorubicin and paclitaxel Using an in vivo Matrigel plug model in rats, antiangiogenic treatment was demonstrated with PFC nanoparticles of doxorubicin and paclitaxel prodrugs targeted to integrins. Therapeutic effects were evaluated using MRI angiogenesis mapping at 3T with paramagnetic PFC nanoparticles targeted to αvβ3 integrin (Figure 3). Angiogenesis was reduced by both treatment formulations relative to the control. Similar results were obtained in vivo in the Vx2 tumor model in rabbits using paclitaxel prodrug (FIG. 4). Thus, in contrast to previous studies that showed loss of paclitaxel or doxorubicin during in vitro elution, the phospholipid prodrug form was retained in the circulation, delivered to the target cell, released with the enzyme, and intended Demonstrated anti-proliferative effect.

Example 3 Synthesis of Sn-2 doxorubicin prodrug
More than 3 sites are available for functional modification of doxorubicin (Figure 5): side chain carbonyl group (site 1), side chain alcoholic group (site 2) and amine functionality in the sugar moiety (site 3 ). In this example, Site-3 was used for the modifications described below.

Reaction of 16: 0-09: 0 ALDO (PC) (1-palmitoyl-2- (9'-oxo-nonanoyl) -glycero-3-phosphocholine) with doxorubicin in methanol (anhydrous) in the presence of TFA As a result, PC doxorubicin (PC-DXR) conjugate 1 was synthesized. The resulting imine was subsequently reduced with sodium cyanoborohydride to produce the final product. The structure of the compound was characterized by ¹ H NMR (400 MHz) spectroscopy (FIG. 7). Proof of concept for this prodrug approach is microscopically demonstrated by the marked inhibition of endothelial cell proliferation caused by intracellularly distributed doxorubicin and the enzyme-released drug described in Example 1 above. It was done.

Example 4 Synthesis of paclitaxel prodrug Paclitaxel prodrug was synthesized according to a 5-step synthesis method and purified by column chromatography (FIG. 8). Briefly, commercial paclitaxel (Avachem Scientific, Inc.) was treated with succinic anhydride in the presence of pyridine to give 2'-succinyl paclitaxel. This intermediate NHS ester (6) may be obtained by reaction with N-succinimidyl diphenyl phosphate (SDPP). The ester was treated with an excess of mono-Boc-ethylenediamine at low temperature, followed by deprotection of tertiary-Boc. A flexible chain diamine (1,3-diaminopropane) was selected to reduce steric hindrance. Finally, paclitaxelamine was subjected to reductive amination with ALDO PC (or PE) to yield paclitaxel prodrug (7).

Example 5. Sn-2 paclitaxel prodrug assay
2F2B mouse endothelial cells (ATCC, Manassas, VA, USA) were cultured in medium for 2 days and up-regulated with 10 nM nicotine or 10 μM angiotensin II to express αvβ3 integrin. The cells may then be exposed to untargeted paclitaxel vs. integrin-targeted GNB nanoparticle treatment of untargeted paclitaxel at varying drug loads (0.5-5 mol%). As a control, cells were exposed to a corresponding amount of free drug for 30 minutes. Nanoparticles released from the wells or unabsorbed drug were washed away. The culture was then grown for 6 days and the number of viable cells attached was counted. When treated with free taxol, nanoparticles targeted to αvβ3 integrin alone or paclitaxel PC prodrug nanoparticles (PC-PTXL) relative to the corresponding amount of saline, the number of cells was significantly reduced (Fig. 9).

Example 6 Synthesis of Cisplatin Prodrugs Two approaches may be followed for the synthesis of platinum-based anticancer prodrugs. The first approach (FIG. 10) may involve the preparation of amine-terminated cisplatin (9) followed by conjugation with oxidized lipids. The coupling intermediate resulting from the amidation reaction of mono Boc-ethylenediamine and compound 8 in the presence of HATU / DIPEA may be subjected to deprotection to yield compound 9. Compound 9 may undergo reductive amination with ALDO PC in methanol to generate cisplatin prodrug-1 (10).

The second approach (FIG. 11) may involve the synthesis of analogs with hydrophobically modified chelating diamines. Cisplatin intermediate 16 may be obtained from compound 13 in three steps. Intermediate 16 may be subjected to complexation with K ₂ PtCl ₄ by maintaining the pH of the resulting solution at pH 6-7. Compound 13 may undergo reductive amination with ALDO PC in methanol to generate cisplatin prodrug-2 (18).

Example 7 Synthesis of methotrexate prodrugs Synthesis of methotrexate conjugates is described in FIG. A short ethylenediamine spacer may be introduced between methotrexate and oxidized lipid. 6-Bromomethyl-pteridine-2,4-diamine trihydrobromide (BPT · HBr, 23) may be purchased from Ube Industries and coupled with intermediate 24 to produce 25. Compound 25 is deprotected from tertiary-Boc to yield an amine-terminated methotrexate (26) and 26 ALDO (PE which may be followed by ester hydrolysis to yield a methotrexate prodrug (27). ) Or (PC) may be carried out as described above.

Example 8. Synthesis of Bortezomib Prodrug The asymmetric synthesis of Bortezomib prodrug (FIG. 13) may involve the preparation of Intermediate-1 (N-sulfinyl α-aminoboron pinacolato complex) by the following published method. Selective removal of the N-sulfinyl group under mild acidic conditions can result in amine hydrochloride (Intermediate 2). Intermediate 2 may then be coupled with N-Boc-L-phenylalanine by a reaction protocol mediated by TBTU / DIPEA. Intermediate-3 (amine hydrochloride) can then be coupled with commercially available 3-aminopyrazine-2-carboxylic acid to produce bortezomib pinacol boronate. This may subsequently be hydrolyzed under two-phase conditions utilizing isobutylboronic acid as a pinacol sequestering agent. Finally, Intermediate-4 can undergo reductive amination mediated by sodium cyanoborohydride with ALDO (PC) in the presence of catalytic TFA to produce bortezomib prodrug.

Example 9. Synthesis of MYC-inhibitor prodrug
Myc-inhibitor-1 may be synthesized by reacting 4-ethylbenzaldehyde with rhodanine in the presence of catalytic Tween-80 in potassium carbonate solution at ambient temperature (FIG. 14). The mixture may be neutralized with 5% HCl. The precipitant may then be treated with saturated NaHSO ₃ and recrystallized with aqueous ethanol. The rhodanine derivative may be reacted with piperidine-mono-tertiary Boc and formaldehyde. Deprotection of tertiary Boc in the presence of TFA can produce Myc-inhibitor-1. Finally, Myc-inhibitor-1 can undergo reductive amination mediated by sodium cyanoborohydride with ALDO (PC) in the presence of catalytic TFA to produce a Myc-rhodanine prodrug.

Example 10 Myc-inhibitor prodrug in restenosis
Myc encodes a helix-loop-helix transcription factor that is up-regulated in 50-80% of human cancers and is implicated in 100,000 US cancer deaths per year. Myc heterodimerizes with its partner Max to control target gene transcription and is deeply integrated into regulatory and management mechanisms that govern cell viability and proliferation. Recent assessments suggest that Myc binds to approximately 25,000 sites in the human genome. Loss of Myc protein inhibits cell growth and growth, accelerates differentiation, increases cell adhesion, and enhances the response to DNA damage.

  We are the ideal target for MM where Myc allows anticancer therapy, especially Myc-Mad interaction, while it is highly overexpressed by selective and destructive interference of Myc-Max dimerization I believe there is.

  FIG. 27 illustrates that particles targeted to αvβ3 comprising a myc prodrug reduces SMC proliferation. Human coronary artery smooth muscle cells were placed on coverslips (2500 cells) and cultured for 2 hours. The treatment was repeated 6 times each. Intramural delivery of αvβ3-targeted particles comprising a myc prodrug, either alone or together with a stent, provides an attractive new approach to restenosis.

Example 11 Synthesis of Lenalidomide Prodrug Lenalidomide was subjected to reductive amination in the presence of ALDO (PE) or (PC) in methanol (anhydrous) in the presence of TFA, followed by one-pot reduction with NaCNBH _3. Produces a lenalidomide prodrug (FIG. 15).

Example 12 Synthesis of fumagillin prodrug The synthesis of the first prodrug (fumagillin prodrug 1) was performed in a straightforward manner by saponification of fumagillin to fumagillol dicyclohexylamine salt, which was then converted to DCC / Activation was performed using a DMAP-mediated carbodiimide coupling protocol. And then derivatized together with oxidized lipids.

(3R, 4S, 5S, 6R) -5-Methoxy-4-((2R, 3R) -2-methyl-3- (3-methylbut-2-enyl) oxiran-2-yl) -1-oxaspiro [2.5 ] Octano-6-ol (fumagillol, 2).
Fumagillin dicyclohexylamine salt (0.2 g, 0.31 mmol) was suspended in 2 mL of 1: 1 methanol: water and treated with 0.07 mL of 35% NaOH solution (0.62 mmol). The dark brown mixture was stirred in an ice bath for 2 hours, then warmed to room temperature and treated with the same equivalent of NaOH solution. The mixture was stirred until no starting material was detected by TLC (about 4 h); methanol was evaporated and the residue was extracted with ethyl acetate. The mixture was then extracted with 5% citric acid, brine, bicarbonate and again with brine, then dried over MgSO ₄ and concentrated in vacuo. The crude product was further purified by treatment with activated charcoal in acetonitrile and filtered through a celite pad. Concentration gave 59 mg of colorless solid (70%). Obtained HRMS: MH + (283.3).

General procedure for the preparation of fumagillin prodrug 1:
C16-09: 0 (COOH) PC 1-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (3.2 mmol) solution followed by (DMAP (44 mg, 3.2 mmol) and DCC (46 mg, 3.2 mmol) Was added to a solution of fumagillol (11 mg, 0.4 mmol) in dry dichloromethane (1 ml) The reaction was stirred overnight at room temperature and the mixture was then stirred in EtOAc / n hexane (1: 2, v / v). ) Was passed over a short pad of silica gel, and the filtered solvent was removed in vacuo, leaving an oil residue that was eluted with EtOAc / n hexane (1: 4, v / v as the eluting solvent). Purification by column chromatography on SiO ₂ in v) gave the fumagillin prodrug-1 compound as a pale yellow solid, observed HR-MS: 991.5475 (M + 2K-2H).

Other strategies and specific examples:
The challenge of this process is to develop fumagillin and its associated candidate optically and chemically robust analogs with improved membrane retention properties. One of the strategies is to introduce similar parts hydrophobically throughout the system, following the removal of unstable, pharmacologically inactive (and irrelevant) functionality from their structure To do. In the present invention, we intend to replace all trans-decatetraenedioates with photostable, non-bonded unsaturated chains. We synthesize fumagillin analogs in which each of the potentially reactive epoxy groups is substituted individually or in combination. Ester linkages are replaced by stable covalent incorporation. A parallel synthesis strategy allows the incorporation of moieties that provide the film attachment properties of these analogs. The decatetraendioic tail is replaced with a hydrophobically comparable moiety. Synthetic methodologies for making fumagillin analogs have been explored by several groups and disclosed in the literature. We develop an efficient synthetic methodology for proposed analogs for further study on detailed testing of its biological activity with significant therapeutic potential. (See Figure 34.)

Synthesis of Stable Fumagillin Prodrug Fumagillin Prodrug 1: Fumagillol (1) is produced by hydrolysis of fumagillin and with commercial PAzPC (16: 0-9: 0 COOH PC) mediated by DCC Attached to the coupling. The product is recovered and immediately subjected to reaction with LiCl in the presence of acetic acid to open the spiroepoxide with chloromethyl functionality.

Fumagillin prodrug 2: Fumagillol is activated with bis (4-nitrophenyl) carbonate and subsequently reacted with monobok-ethylenediamine to produce 6. In a typical experimental procedure, a mixture of fumagillol, bis (4-nitrophenyl) carbonate and DMAP in dry CH ₂ Cl ₂ is stirred for 7 hours under N ₂ atmosphere. The reaction is diluted with CH ₂ Cl ₂ and washed with H ₂ O. The organic layer is dried over MgSO ₄ and concentrated. Flash chromatography (EtOAc-hexane) is used to give the activated fumagillol. Monobock protected ethylenediamine is then coupled to prepare Intermediate-6. The product is recovered and immediately reacted with LiCl in the presence of acetic acid to open the spiroepoxide with chloromethyl functionality. Bock is deprotected with TFA. The product is then subjected to reductive amination mediated by sodium cyanoborohydride in the presence of ALDO PC.

Synthesis of precursor compounds: Ovalicin (4) is synthesized according to literature procedures by Takahashi et al. ^40a Commercially available 2,3: 5,6-di-O-isopropylidene-RD mannofuranose is used as starting material to produce optically pure ovalicin in 10% overall yield. Fumagillol is purchased from Sinova, Inc. and used as is.

スキーム1．オバリシン及びフマギノンの前駆物質から類縁体1-2の合成 Fumagillol may be subjected to oxidation using pyridinium chromium chloride / pyridine (PCC) to produce fumaginone (3). In a typical experimental procedure, a mixture of fumagillol (1 eq), pyridinium chromium chloride (PCC, 8 eq) and pyridine (5 eq) in anhydrous CH ₂ Cl ₂ at ambient temperature is allowed to stir for 10 hours. The completion of the reaction is then monitored by thin layer chromatography. The reaction is purified by silica gel column chromatography (EtOAc-CH ₂ Cl ₂ ) to give fumaginone as a clear oil.

Scheme 1. Synthesis of analog 1-2 from the precursors of ovalicin and fumaginone

スキーム2．オバリシン及びフマギノンの前駆体から類縁体3-6の合成 Synthetic Analogue 1-2: As briefly discussed above, in the present invention we are all light-stable and non-bonded unsaturated chains (analogue 1-2) all trans-decatetraenedioates Is intended to replace Thus, analogs 1-2 with various unbonded substituents with hydrophobic tails are prepared with either hydrogen (fumagillin series) or hydroxyl groups (Ovalicin series). Briefly, the precursor compounds are subjected to indium-mediated organometallic allylation with each substituted allyl bromide.

Scheme 2. Synthesis of Analog 3-6 from Ovalicin and Fumaginone Precursor

Analog 3-6: Analog 3 can be synthesized from ovalicin hydrazone. Briefly, ovalicin (or fumaginone) is treated with LiCl in the presence of acetic acid to open the spiroepoxide with chloromethyl functionality. A solution of ovalicin in THF is stirred with LiCl and acetic acid for 36 hours, and after the removal of THF, chloroform is subsequently added. The organic layer is washed with H ₂ O, sodium bicarbonate, dried and purified by silica chromatography (30% EtOAc-hexane).

This intermediate is then treated with hydrazine under mildly acidic (acetic acid) conditions to give the corresponding hydrazone. The hydrazone is then subjected to selective reduction with sodium cyanoborohydride followed by DCC / DMAP mediated amidation in the presence of the corresponding acid (E-isogeranoic acid) ^40e (Scheme 2). In the final step, spiroepoxide functionality is optionally modified. Analog 3 is treated with KOtBu in THF at 0 ° C. to provide a modified spiro epoxide moiety containing analog-4.

  In a separate route, intermediate-1 is treated with hydroxylamine in the presence of a mild acid to give the corresponding oxime derivative. The oxime derivative is reduced and subsequently undergoes DCC-mediated esterification with E-isogeranoic acid to produce analog 5. 2,6-octadienoic acid chloride may also be used to esterify the oxime (analog 6).

スキーム3．オバリシン及びフマギノンの前駆体から類縁体7-9の合成 E-isogeranic acid is synthesized from E-isogeraniol according to literature reports by Eustache and co-authors. ^40e E-isogeraniol is subjected to chromium trioxide-mediated oxidation in the presence of sulfuric acid in a mixture of water and acetone to obtain the desired E-isogeranic acid. The acid is obtained in pure enantioselective E-form and is separated from by-products by passing through a silica column. In a simple manner, 2,6-octadienoic acid- (2E, 6E) is converted to the acid chloride by treatment with PCl ₅ . ⁴¹

Scheme 3. Synthesis of Analog 7-9 from Ovalicin and Fumaginone Precursor

Analog 7-9: Based on previous reports by Liu et al. ⁴² , a compound possessing a rotatable single bond between the C10 and C20 carbons on the C4 side chain had fixed epoxide and diene side chains. It showed reduced activity against MetAP-2 compared to analogs. Furthermore, crystallographic data showed that the space important for further optimization is in the pocket ⁴² . All at the same time we decided to replace the acid labile side chain epoxide with a diene moiety.

スキーム 4．オバリシン及びフマギノンの前駆体から類縁体10-11の合成 In the procedure of a typical experiment, analogs -5 is treated with WCl ₆ in the presence of n-BuLi at -78 ° C., and processed by KOtBu in THF continued at 0 ° C., gives the analog 7 (Scheme 3). Under an argon atmosphere, n-BuLi is added dropwise to a solution of WCl ₆ in THF at −78 ° C., followed by continued stirring for 1/2 hour. A solution of analog-5 is added and allowed to react for 2 hours. NaOH is added to the reaction mixture. The product is then extracted with ether. The extract was washed with NaOH and water. The residue was purified by column chromatography (ethyl acetate hexane) to give analog-7. Analogs 8-9 are synthesized from analogs 4 and 6 respectively, following the route described above for the synthesis of analog-7.

Scheme 4. Synthesis of analog 10-11 from the precursors of ovalicin and fumaginone

ダイヤグラム 4．オバリシン及びフマギノンの前駆体から類縁体12-15の合成 Analog 10-11: Furthermore, we replace exocyclic side chain epoxides with oxime functionality that may provide the desired geometry and substitution pattern. Previous reports by Pyun et ^40m showed that exhibit activity (approximately 1 nM in MetAP-2 analysis) excellent benzyl oxime with analog ^40m. C4 oxime analogs are synthesized and evaluated. As previously reported by Fardis et al., ^40b , Intermediate 3 can be readily synthesized from fumagillol. Intermediate-3 is subjected to ozonolysis at -78 ° C followed by treatment with dimethyl sulfide to give the corresponding ketone derivative. The ketone derivative is treated with aminobenzyl oxime (H ₂ N—OBn) in the presence of TsOH and molecular sieves (4Å) to produce a mixture of E and Z benzyl oximes (intermediate 4). Intermediate-4 is then oxidized to the corresponding ketone derivative. Treatment of intermediate-4 with hydrazine or hydroxylamine generates the corresponding oxime derivative. It is then reduced and activated with DCC / DMAP to react with E-isogeranoic acid to produce analog 10-11.

Diagram 4． Synthesis of Analog 12-15 from Ovalicin and Fumaginone Precursors

ダイヤグラム 5．オバリシン及びフマギノンの前駆物質から類縁体16-19の合成 Analogs 12-15: Fumaginoru, without combining the MetAP2 covalently, a novel synthetic analogues of fumagillin which has been shown to selectively inhibit MetAP2 and endothelial cell proliferation ^{40 g.} Fumaranol consists of a bicyclic ring structure with a spiro epoxy part open upward. We decided to explore related fumaranol analogs in line with the strategy discussed above. To form a carbanion at the R-position of the 6-ketone group, KOH and analog-10 are reacted, and the carbanion undergoes an SN2-type reaction in the molecule to open the spiro epoxy group (Diagram 4). As a result, a bicyclic ring structure (analog 12) is produced. Similarly, analogs 13-15 are synthesized from analogs 7 and 8, respectively.

Diagram 5. Synthesis of Analog 16-19 from Ovalicin and Fumaginone Precursors

スキーム 5．オバリシン及びフマギノンの前駆体から類縁体-20の合成

スキーム 6．フマギロールから類縁体-21の合成 Analogs 16-19: We further attempt to synthesize analogs in which the spiro (C-3) epoxide is opened with a thiomethoxide (Scheme 5) functionality. Briefly, thiomethoxide is added at room temperature to a stirred solution of analog-7 in anhydrous DMF. The reaction is stirred for 2 hours, then diluted with ethyl acetate and washed with saturated aqueous NaHCO ₃ and water. The organic phase is dried (anhydrous Na ₂ SO ₄ ) and the residue is purified by column chromatography on silica gel (ethyl acetate: hexane) to produce analog-16. Following similar methodology, analogs 17-19 are synthesized from analogs 8-9 and 4, respectively.

Scheme 5. Synthesis of analog-20 from the precursors of ovalicin and fumaginone

Scheme 6. Synthesis of analog-21 from fumagillol

ダイヤグラム 6．提案されたフマギリン類縁体22-27の構造 Synthetic analogs 20-25 (fumagillin-PAF analogs): As previously described, one of the challenges of this process is to develop new fumagillin analogs with improved membrane attachment properties. is there. Towards this goal, a series of fumagillin-PAF analogs targeted to understand the tolerance of MetAP2 to substitutions at C4 and C6 are synthesized. First, C6 is modified by keeping the C4 side chain unchanged. Placing platelet activating factor (PAF) -lipid (16: 0) at C6 is expected to improve the membrane adhesion properties of these compounds. We plan to use short (glycine) and intermediate (ε-aminocaproic acid) spacers as the flexible part of the analogs that give a connection between the cyclic carbon and the hydrophobic tail (Schemes 5 and 6). To do. The linker molecule may play an important part in driving the enzymatic hydrolysis of fumagillin candidates linked to PAF. Effect of linker length in the degeneration of phospholipid prodrugs, was studied by the previously Dahan et ^28f. The shorter linker (2-carbon) was found to cause 20-fold less prodrug release compared to the 5-carbon linker ^28f . Briefly, ovalicin (or fumaginone) is treated in the presence of ε-aminocaproic acid under mildly acidic (acetic acid) conditions to give the corresponding hydrazone. The hydrazone is selectively reduced with sodium cyanoborohydride and subsequently subjected to DCC / DMAP-mediated amidation in the presence of PAF (16: 0) to give analog-20.

Diagram 6． Structure of the proposed fumagillin analog 22-27

Instead, fumagillol is activated with bis (4-nitrophenyl) carbonate and subsequently reacts with glycine. In a typical experimental procedure, a mixture of fumagillol, bis (4-nitrophenyl) carbonate and DMAP in dry CH ₂ Cl ₂ is stirred for 7 hours under N ₂ atmosphere. Dilute the reaction with CH ₂ Cl ₂ and wash with H ₂ O. The organic layer is dried over MgSO ₄ and concentrated. Flash chromatography (EtOAc hexane) gave activated fumagillol. Glycine is then linked to prepare intermediate-6. Briefly, a solution of activated fumagillol, glycine and DMAP in dry CH ₂ Cl ₂ under N ₂ atmosphere is stirred for 2 hours. The completion of the reaction is monitored by thin layer chromatography. The reaction is worked up by diluting with CH ₂ Cl ₂ and washing the organic layer with H ₂ O. Flash chromatography (MeOH-EtOAc) following removal of the solvent resulted in the production of Intermediate-6. Finally, carbodiimide-mediated esterification (DCC / DMAP) of intermediate-6 with PAF (16: 0) agent produces analog 21.

After replacing the PAF agent with C6, C4 side chain modification of fumagillin is performed to develop a MetAP-2 inhibitor with desirable pharmacological properties. The spiroepoxide rings of analogs 18 and 19 are opened with thiomethoxide to produce analogs 23 and 25.
Replacement of the C4 side chain with benzyl oxime and diene side chains is accomplished to give analogs 22, 24, 26-27 (Diagram 6).

Characterization of synthetic fumagillin analogs: Detailed characterization of synthetic fumagillin analogs is performed by NMR, FT-IR, mass spectrometry and elemental analysis. The signal assignments of ¹ H and ¹³ C and the spatial proximity of protons are based on COZY, NOE experiments. ¹ H and ¹³ C signal assignments are made based on COZY, NOESY and selective 1DTOCSY13 measurements. The proton-proton coupling constant of the aliphatic part of the molecule is determined by a first order approximation from the ¹ H-NMR spectrum after Gaussian apodization for improved resolution. Studies of the cyclohexyl ring and the C-4 side chain are performed in a similar manner.

Example 13. Fumagillin Prodrug Efficacy As shown in FIG. 19, fumagillin dissolved in the lipid membrane is rapidly released in vivo, making it virtually ineffective in vivo. However, FIG. 20 shows the in vivo efficacy of fumagillin prodrug administered with the nanoparticles of the present invention. In particular, the figure shows in vivo MR signal increase after treatment with targeted fumagillin nanoparticles (ab) and control (drug pear, cd); nano targeted to ανβ3 integrin with 2.28% fumagillin Particles, nanoparticles targeted to ανβ3 integrin without drug, untargeted nanoparticles with 2.28 mole% fumagillin PD, vs. ανβ3 integrin with 2.28 mole% fumagillin PD Reduced Matrigel implantation volume (%) in rats treated with nanoparticles.

  FIG. 21 shows the effect of fumagillin prodrug on in vitro cell proliferation analysis. Left panel shows nanoparticles incorporated with fumagillin prodrug on human umbilical vein endothelial cells (HUVEC) and control nanoparticles (targeted drug pear, target) for cell proliferation by CyQuant NF analysis And the effect of untargeted and targeted fumagillin). The right panel shows the cellular metabolic activity by Alamar Blue analysis.

Example 14 Docetacel Prodrug A docetacel prodrug synthesized as shown in FIG. 18A or B was administered to 2F2B cells in alamar blue analysis to measure metabolic activity. FIG. 22 shows a comparison between αvβ3 targeted particles without docetacel and αvβ3 targeted particles with docetacell and αvβ3 targeted particles with docetacel prodrug. As the analysis progressed, particles comprising a prodrug demonstrated sustained suppression of cellular metabolic activity as compared to the targeted control of docetacel pear.

Example 15. Synthesis of Photodynamic Therapy (PDT) Prodrugs In a typical procedure, zinc-chelated porphyrin-based PDT prodrugs are synthesized in a linear manner. The amine-terminated zinc porphyrin PDT prodrug is then subjected to reductive amination with ALDO PC in the presence of NaBH ₄ and catalytic trifluoroacetic acid.

Example 16 Polysorbate micelles This application encompasses the design, synthesis, physicochemical characterization, and use of novel nanoparticulate systems for diagnostic therapeutic applications and gene delivery systems. Multifunctional nanoparticles play a very important role in anticancer drug delivery. As new and new applications are constantly being developed, the possibilities for nanoparticles in cancer drug delivery are endless. Cancer has physiological barriers such as vascular endothelial pores, heterogeneous blood supply, heterogeneous configurations, and the like. Overcoming these physiological barriers is critical for successful therapy. For cancer detection and treatment using targeted molecular imaging and drug delivery, nanoparticulate drugs are the latest achievement in the medical field and form the basis of nanomedical research. A variety of nanodevices have been explored including nanopores, nanotubes, quantum dots (QDs), nanoshells, dendrimers and shell cross-linked nanoparticles, PFC nanoparticles, biodegradable hydrogels, and the like.

Nanoscale devices are 100 times smaller than human cells, but are similar in size to large biomolecules (enzymes and receptors). With respect to size and shape, nanoparticles below 50 nm can easily enter most cells. And as they circulate in the body, anything less than 20 nm can exit the blood vessels. Nanoparticles are designed as vectors that carry a high payload of contrast agents for efficient detection in vivo, saving large amounts of normal cells while saving high amounts of chemotherapeutic agents or therapeutic genes into malignant cells It is more appropriate to serve as a customized targeted drug delivery excipient that carries the dose. In this approach, the nanoparticles significantly reduce or eliminate the often unacceptable side effects associated with many current cancer therapies. Furthermore, the type of nanoparticles is very important and can be classified into two main classes (inorganic and organic). Liposomes, dendrimers, carbon nanotubes, emulsions and other polymeric nanoparticles are examples of a widely studied group of organic particles. Most inorganic nanoparticles share the same basic structure of the central core that defines the protective, organic coating on the surface and the fluorescent, optical, magnetic and electronic properties of the particle. This outer layer protects the core from degradation in a physiologically invasive environment, with positively charged agents and biomolecules with basic functional groups (amines and thiols), electrostatic or covalent bonds, or both Can be formed. In terms of size, inorganic nanoparticles can be produced with very small dimensions ranging from 2-50 nm (gold to iron oxide particles), however, achieving such a small range with organic precursors Is challenging. Larger nanoparticles in cancer can make them difficult to avoid organs (liver, spleen and lung). It is constantly removing foreign substances from the body. Furthermore, they must be able to take advantage of subtle differences in the cells to be distinguished between normal and cancerous tissues. Indeed, it is only recently that researchers have begun to successfully and successfully plan nanoparticles that can effectively bypass the immune system and can actively target tumors.

  Thus, despite recent advances in the development of targeted therapeutic imaging nanoparticles, there is a further need for persistence for a stable system that is water soluble with extravascular particles (<20 nm). Exists in. The required features include bioseparability, tunable release characteristics, and internal sequestration of easily biometabolizable therapeutic agents that are resistant to hostile in vivo environments. Based on these requirements, we have developed ultra-fine “flexible nanocellelle nanoparticles for diagnostic therapeutic applications. Nanoparticles are targeted ligands (integrins) specific to target cells or tissues. αvβ3, α5β1, ICAM-1, VCAM-1, VLA-4, etc.) to make them specific for the target cell or tissue.Nanoparticles are also intended for their application in imaging and therapy And may include bioactive substances, prodrugs (fumagillin, myc-max inhibitor, camptothecin, cisplatin, steroids, methotrezate, paclitaxel / docetacel, etc.) radionuclides, etc. Particles are MR, CT, optical, photoacoustic Incorporated with contrast agents for PET, SPECT and US.

Preparation of nanocell nanoparticles We prepared soluble nanocell nanoparticles in water with a nominal hydrodynamic diameter between 16-20 nm. The core of these particles is made of a biologically relevant surfactant (ie Tween 80, span 80, sorbitan sesquioleate, sorbitan monolaurate) with a phospholipid surrounding it. The particles are thoroughly characterized by physicochemical methods.

Typical procedure for the preparation of nanocell particles loaded with avb3 targeted Myc-max inhibitor:
In a typical experimental procedure, a surfactant co-mixture was prepared from high purity egg yolk phosphatidylcholine (99 mol%, 395.5 mg), αvβ3-peptidomimetic antagonist (0.1 mol%, Kereos, Inc, conjugated to PEG2000-phosphatidylethanolamine). St. Louis, MO, USA) (1 mol%, 6.0 mg) and 2 mol% of myc-max prodrug. The surfactant co-mixture was dissolved in anhydrous chloroform, evaporated under reduced pressure to form a film, dried in a 50 ° C. vacuum oven overnight, and dispersed in water (5 mL) by probe sonication. This suspension was combined with polysorbate mixture (20% v / v, 4 ml), distilled and deionized water (77.3% w / v, 15.23 ml total) and glycerin (1.7%, w / v, 0.37). ml). The mixture was continuously post treated with S110 Microfluidics emulsifier (Microfluidics) at 0 ° C. for 4 minutes at 20,000 psi. The nanocell particles were dialyzed against nanopure (0.2 μm) water using a 20,000 Da MWCO cellulose membrane for a long time and then passed through a 0.45 μm Acrodisc Syringe filter. The nanoparticles were typically stored at 4 ° C. under an argon atmosphere to prevent any bacterial growth. The hydrated hydrodynamic diameter of the particles (16 ± 4 nm) is measured by a Brookhaven dynamic light scattering experiment operating the laser at 90 °. The diameter and particle height of the dehydrated state are TEM (negatively stained with 1% uranyl acetate and dried on the nickel surface), and AFM (Figure xx) (tapping mode, drop placed, Measured on a glass cover slip).

In vitro prodrug test: cytotoxicity analysis Human B16 melanoma cells were seeded on 96-well plates (5000 cells / well). After 24 hours, the cells are either avb3-integrin-targeted nanocell myc prodrug (approximately 0.5 uM myc-PD), free myc, targeted drug pear, or untargeted myc prodrug nanocell. Incubated for 1 hour (6 replicates per sample per plate). The wells were washed 3 times with PBS and the plate was returned to the incubator. Cell metabolic activity was measured using Alamar Blue (Invitrogen) 2 to 4 days after drug exposure. This analysis is based on the reduction of non-fluorescent dyes to fluorescent substrates in metabolically active cells. This reduction is typically due to different oxidoreductase enzyme systems that use NAD (P) H as the primary electron donor. This redox reaction was monitored using a fluorescence plate reader (Ex 570 nm and Em 587 nm) where the resulting signal was proportional to the number of living cells present. The intensity of the signal from each sample was normalized to the signal from a positive control (no drug nanoparticles targeted with αvβ3 integrin) (n = 3-6 plates per time point) (FIG. 28).

Example 17: Prodrug comprising PNA
PNA and conjugates are synthesized on 2 μmol Fmoc-PAL-PEG-PS on an automated peptide synthesizer by standard automated Fmoc PNA synthesis procedure utilizing commercial monomers (Panagene, Korea). According to the final step of automated synthesis, the resin is washed with dry DMF (2 × 3 mL) and dry CH ₂ Cl ₂ (2 × 3 mL) and then dried under N ₂ flow. The resin is then shaken in a vial with trifluoroacetic acid (300 μL) and m-cresol (100 μL) for 2 hours at room temperature to release and deprotect the PNA. The solution is filtered from the resin and added to ice-cold Et ₂ O (5 mL) and held at 4 ° C. for 1 hour. The resulting precipitate is collected by centrifugation and purified by reverse phase HPLC. PNA lysine conjugates are synthesized on an automated peptide synthesizer (2 μmol) using standard Fmoc chemistry. PNA lysine conjugate in DMSO (0.19 μmol determined spectrophotometrically) is added to a solution of PAzPC and prior to the addition of 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide methiodide. Allow to stir for 30 minutes. The resulting solution was allowed to stir for 16 hours and purified by reverse phase HPLC technique.

ダイヤグラム 7．カンプトテシンプロドラッグ1(トップ)およびプロドラッグ2(ボトム)のための合成経路

a.1) カンプトテシンプロドラッグ1の合成：カンプトテシンプロドラッグは、DCC/DMAPを媒介としたカップリングプロトコルが存在する状態でPAzPC(脂肪酸で変性され酸化された脂質16:0-9:0 COOH PC)と化合物の直接接合(カップリング)によって製造される。 Example 18: Synthesis of camptothecin prodrug

Diagram 7. Synthetic routes for camptothecin prodrug 1 (top) and prodrug 2 (bottom)

a.1) Synthesis of camptothecin prodrug 1: Camptothecin prodrug is a PAzPC (fatty acid-modified and oxidized lipid 16: 0-9: 0 COOH PC in the presence of a DCC / DMAP-mediated coupling protocol. ) And a compound (direct coupling).

a.2) Synthesis of camptothecin prodrug 2: Camptothecin is activated with bis (4-nitrophenyl) carbonate and subsequently reacted with monobok-ethylenediamine to produce 6. In a typical experimental procedure, a mixture of camptothecin, bis (4-nitrophenyl) carbonate and DMAP in dry CH ₂ Cl ₂ is stirred for 7 hours under N ₂ atmosphere. The reaction is diluted with CH ₂ Cl ₂ and washed with H ₂ O. The organic layer is dried over MgSO ₄ and concentrated. Flash chromatography (EtOAc-hexane) is used to give the activated fumagillol. The monobock protected ethylenediamine is then coupled to prepare Intermediate-6. The product is recovered and immediately subjected to PAzPC and DCC / DMAP mediated coupling. The chemical identity of both analogs is confirmed by NMR and mass spectrometry.

Example 19. Methylprednisolone prodrug Methylprednisolone prodrug is a direct combination of PAzPC (fatty acid modified and oxidized lipid 16: 0-9: 0 COOH PC) and compounds in the presence of a DCC / DMAP-mediated coupling protocol. Generated by coupling.

Example 20. How to Ligand Ligands to Nanoparticles Ligand attachment to nanoparticles naturally interacts with adamantane on peptides or small molecule ligands to form inclusion complexes Uniquely achieved by following the inclusion compound protocol with β-cyclodextrin (β-CD) on the particles. Briefly, cyclodextrin-PEG-DSPE derivatives are synthesized via mono-6-deoxy-6-amino-β-cyclodextrin. One of the seven major hydroxyl groups of β-cyclodextrin is tosylated using p-toluenesulfonyl chloride. Substitution of the tosyl group with azide and subsequent reduction with triphenylphosphine gives mono-6-deoxy-6-amino-β-cyclodextrin. Carboxyl activated PEG-DSPE is conjugated with mono-6-deoxy-6-amino-β-cyclodextrin to produce cyclodextrin-PEG-DSPE. Adamantanamine is joined directly through a short spacer in solid-phase peptide synthesis to the carboxyl terminus of the peptide to generate an adamantane peptide / ligand. Simple room temperature mixing of adamantaneamine and β-cyclodextrin carrying nanoparticles produces targeted nanoparticles linked to peptides.

Claims (20)

A composition for in vivo delivery of a compound to a target cell, the composition comprising non-liposomal particles and at least one prodrug, wherein
(a) the outer surface of the particle is a membrane comprising at least one prodrug, further comprising about 100% to about 60% phospholipid, and the outer surface of the particle is a leaflet external to the cell membrane of the target cell. In vivo, such fusion results in a prodrug transfer from the particle to the outer leaflet of the target cell,
(b) the prodrug comprises a compound of less than about 3000 da linked to the acyl moiety of the phosphoglyceride, wherein the compound may be released from the phosphoglyceride backbone by enzymatic cleavage;
(c) the prodrug essentially remains on the outer surface cell membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell, and
(d) A composition wherein the prodrug is further transported from the outer leaflet to the inner leaflet of the cell membrane of the target cell, resulting in the release of the compound intracellularly by cleavage of the enzyme-cleaving linkage.   2. The composition of claim 1, wherein the outer surface of the particles comprises at most 3% of the prodrug.   2. The composition of claim 1, wherein the outer surface of the particles comprises at most about 10% of polysorbate.   2. The composition of claim 1, wherein less than about 10% of the outer surface is crosslinked.   The composition of claim 1, wherein the outer surface further comprises a homing ligand.   6. The composition of claim 5, wherein the outer surface is not PEGylated except for the homing ligand.   6. The composition according to claim 5, wherein the homing ligand is selected from the following group: antibody, antibody fragment, peptide, asialoglycoprotein, polysaccharide, nucleic acid, small molecule and peptidomimetic.   2. The composition of claim 1, wherein the compound is linked to the sn-2 acyl moiety of the phosphoglyceride.   9. The composition of claim 8, wherein the acyl portion of the prodrug is at least 4 carbon atoms in length from the glycerin backbone sn-2 ester linkage.   2. The composition of claim 1, wherein the prodrug comprises a compound selected from the group consisting of peptides, peptidomimetics or analogs, organometallic complexes, organic molecules, nucleic acids or analogs thereof.   2. The composition of claim 1, wherein the enzyme-cleaving linkage is not substantially cleaved extracellularly in vivo.   The prodrug is a compound selected from the group consisting of compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII and XIX The composition of claim 1 comprising:   2. The composition of claim 1, wherein the prodrug comprises a compound selected from doxorubicin, docetacel, methylprednisolone, fumagillin or an analog thereof, camptothecin or an analog thereof and a PDT drug.   2. The composition of claim 1, wherein the prodrug comprises a phosphoglyceride selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidylserine.   2. The composition of claim 1, wherein the particles are between about 10 nm and 10 micrometers.   2. The composition of claim 1, wherein the compound is a prodrug. A method for in vivo delivery of a compound to a target cell, the method comprising administering the composition to a subject, wherein the composition pharmaceutically comprises a non-liposomal particle and at least one prodrug. In an acceptable carrier comprising:
here,
(a) the outer surface of the particle is a membrane comprising at least one prodrug, further comprising about 100% to about 60% phospholipid, and the outer surface of the particle is a leaflet external to the cell membrane of the target cell. In vivo, and such fusion results in a prodrug transfer from the particle to the outer leaflet of the target cell,
(b) the prodrug comprises a compound of less than about 3000 da linked to the acyl moiety of the phosphoglyceride, wherein the compound may be released from the phosphoglyceride backbone by enzymatic cleavage;
(c) the prodrug essentially remains on the outer surface cell membrane of the particle until transfer of the prodrug to the outer leaflet of the target cell
(d) The prodrug is further transported from the outer leaflet to the inner leaflet of the target cell's cell membrane, resulting in the release of the compound intracellularly by cleavage of the enzyme-cleaving linkage.   18. The method of claim 17, wherein in the method, the composition is administered intravenously.   18. The method of claim 17, wherein in the method, the composition further comprises an additional active pharmaceutically active ingredient.   In the above method, the composition comprises the group consisting of compounds I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII and XIX 18. The method of claim 17, comprising a prodrug selected from

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